The anterior cruciate ligament (ACL) is the most frequently injured knee ligament, with approximately 75,000 ligament surgeries performed annually in the United States. The autologous bone-patellar tendon- bone graft and the hamstring tendon graft are the most commonly utilized grafts for ACL reconstruction surgeries. In the early post-operative healing period, the site of graft contact in the bone tunnels represents the weakest point mechanically. Therefore, the clinical success of ACL reconstructive surgeries depends on graft fixation and the formation of a functional interface between bone and soft tissue. The native ACL-bone insertion zone is comprised of four different regions: ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone. Our approach to engineering a functional interface is through the novel co-culture of osteoblasts and ligament fibroblasts on a multi-phased scaffold system with a gradient of structural and functional properties that mimic those of the native insertion zones. These design parameters are chosen because multiple types of cells and an inherent increase in mineral content are both observed across the insertion zone. Our hypothesis is that the co-culture of osteoblasts and ligament fibroblasts on a multi-phased scaffold with a biomimetic calcium phosphate (Ca-P) composition will result in the formation of a fibrocartilage-like interracial zone in the scaffold. To test this hypothesis, the specific aims of this proposal are: Aim 1. To characterize the mineral content (Ca-P distribution, Ca/P ratio) across the native ACL-bone interface. Aim 2. To develop a multi-phased scaffold with a biomimetic compositional variation of Ca-P and to examine the effects of osteoblast-ligament fibroblast co-culture on the development of interfacial zone specific markers (proteoglycan, types II & X collagen) on this scaffold. The global hypothesis of our approach is that the compositional and structural distributions in the native tissue are correlated with functionality. The proposed evaluation of the ACL-bone interface will provide previously unavailable insight into the organizational characteristics at the insertion zone, and identify design objectives for scaffolds with controlled inhomogeneity. By implementing both osteoblast- fibroblast co-culture and the appropriate zonal dependent variations in mineral content into the design of multi-phased scaffolds, we seek to lay the foundation for the development of functional biomimetic scaffolds which can promote healing and facilitate soft tissue graft and bone integration in vivo.